WO2010090359A1 - Novel l-arabinitol dehydrogenase, and method for producing l-ribulose using same - Google Patents

Novel l-arabinitol dehydrogenase, and method for producing l-ribulose using same Download PDF

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WO2010090359A1
WO2010090359A1 PCT/KR2009/000544 KR2009000544W WO2010090359A1 WO 2010090359 A1 WO2010090359 A1 WO 2010090359A1 KR 2009000544 W KR2009000544 W KR 2009000544W WO 2010090359 A1 WO2010090359 A1 WO 2010090359A1
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arabinitol
dehydrogenase
arabinitol dehydrogenase
ribulose
gene
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PCT/KR2009/000544
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French (fr)
Korean (ko)
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이정걸
문희정
마니시티와리
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건국대학교 산학협력단
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    • C—CHEMISTRY; METALLURGY
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    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/02—Monosaccharides

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  • the present invention relates to a novel L-arabinitol dehydrogenase and a method for preparing L-ribulose using the same, and more particularly, to L-arabinitol dehydrogenase, a nucleic acid molecule encoding the same, and the nucleic acid molecule. It relates to a vector, a transformant comprising the vector and a method for producing L-ribulose using the L- arabinitol dehydrogenase.
  • the present invention is derived from a study carried out as part of the microbial genome utilization technology development project of the Ministry of Education, Science and Technology [Task No. 2007-A002-0065, Task name: Development of customized oxidoreductase by in vitro coevolution].
  • L-Ribose is the starting material for the synthesis of drugs per many L-form nucleic acids.
  • L-Ribose has been produced mainly by chemical synthesis from L-arabinose, L-xylose, D-glucose, D-galactose, D-ribose or D-mannono-1,4-lactone.
  • chemical synthesis has disadvantages such as low total yield, many chemical reaction steps, complex purification process and by-product formation.
  • biological L-ribose preparation from ribitol or L-ribulose has been studied.
  • L-ribose is an important key pentose sugar that constitutes the backbone in the synthesis of L-ribonucleosides, L-oligoribonucleosides and many other therapeutic agents.
  • L-nucleoside is a high potential candidate that can be used as a therapeutic material because it has high stability against attack of nucleases and the like in the body as compared to D-nucleoside.
  • L-ribose is known to be highly useful as a raw material for manufacturing medicines such as antiviral drugs and anticancer drugs.
  • the present invention solves the above problems, and the first object of the present invention is to provide a gene of L-arabinitol dehydrogenase.
  • a third object of the present invention is to provide a recombinant expression vector containing the gene of the L- arabinitol dehydrogenase.
  • a fifth object of the present invention is to provide a recombinant L-arabinitol dehydrogenase using transformed recombinant E. coli.
  • a sixth object of the present invention is to provide a method for preparing L-ribulose from L-arabinitol using the enzyme.
  • the present invention is characterized by using the L- arabinitol dehydrogenase of the Debaromyces hansenii strain in the production method of L-ribulose.
  • the present invention is characterized by cloning the L- arabinitol dehydrogenase gene from devaromysis Hanseni through Southern hybridization and colony hybridization.
  • the present invention provides an L-arabinitol dehydrogenase having an amino acid sequence of SEQ ID NO: 4 or a functional fragment thereof.
  • the L-arabinitol dehydrogenase is preferably derived from debaromysis hanseni, but is not limited thereto.
  • the arabinitol dehydrogenase is characterized in that it is specific to L- arabinitol.
  • the molecular weight of the enzyme of the present invention is characterized in that 38 kDa.
  • the enzyme of the present invention is characterized by an increase in activity in the presence of 1 mM Mg 2+ or 1 mM NAD + .
  • the present invention also provides a L- arabinitol dehydrogenase gene encoding the enzyme of the present invention.
  • the gene of the present invention preferably has a nucleotide sequence of SEQ ID NO: 3, at least 85%, preferably at least 90% or more, more preferably from the sequence of SEQ ID NO: 3 in consideration of degeneracy of the genetic code, etc. Is preferably a sequence having at least 95% homology, but is not limited thereto.
  • the present invention provides a method for producing L- arabinitol dehydrogenase by culturing a strain transformed with a recombinant expression vector comprising the L- arabinitol dehydrogenase gene of the present invention.
  • the present invention also provides a method for preparing L-ribulose from L-arabitol using the L-aravinitol dehydrogenase of the present invention.
  • L-arabinitol dehydrogenase of the present invention is characterized by having an amino acid sequence represented by SEQ ID NO: 4.
  • at least one amino acid sequence of SEQ ID NO: 4 may be deleted, substituted, or added to one or more amino acids within a range in which the L-arabinitol dehydrogenase activity indicated by the protein having these amino acid sequences is not impaired.
  • Mutant L-arabinitol dehydrogenase into which a mutation of is introduced is also included in the L-arabinitol dehydrogenase according to the present invention.
  • the present invention includes an L-arabinitol dehydrogenase gene encoding an L-arabinitol dehydrogenase having an amino acid sequence of SEQ ID NO: 4, and the gene sequence represented by SEQ ID NO: 3 may be mentioned. have.
  • the L-arabinitol dehydrogenase gene encoding the above-mentioned variant L-arabinitol dehydrogenase obtained by mutating the nucleotide sequences of SEQ ID NO: 3 is also L-arabinitol dehydrogenation according to the present invention. It is included in the enzyme gene.
  • the present invention includes a recombinant vector containing the L-arabinitol dehydrogenase gene and a transformant transformed by the recombinant vector.
  • the present invention also includes a method for producing L-arabinitol dehydrogenase, wherein the transformant is cultured to separate L-arabinitol dehydrogenase from the culture obtained.
  • L-arabinitol dehydrogenase gene of the present invention is isolated from the cells of devaromysis Hanseni. First, chromosomal DNA is obtained from a strain having a L-arabinitol dehydrogenase gene.
  • PCR polymerase chain reaction
  • PCR amplification fragments are labeled with appropriate reagents, and colony hybridization is performed on the chromosomal DNA library to select L-arabinitol dehydrogenase genes (Current Protocols in Molecular Biology, Vol. 1, 603). Page, 1994).
  • DNA fragments containing the L-arabinitol dehydrogenase gene were recovered by recovering the plasmid from the Escherichia coli selected by the above method using alkaline protocols (Current Protocols in Molecular Biology, Vol. 1, p. 161, 1994). You can get it. After determining the nucleotide sequence by the above method, it is possible to obtain the entire gene of the present invention by hybridizing the DNA fragment prepared by digestion by restriction enzymes of the DNA fragment having the nucleotide sequence as a probe.
  • SEQ ID NO: 3 shows the nucleotide sequence of the L- arabinitol dehydrogenase gene of the present invention
  • SEQ ID NO: 4 shows the amino acid sequence encoded by the gene.
  • the transformed microorganism of the present invention is obtained by introducing the recombinant vector of the present invention into a host suitable for the expression vector used when producing the recombinant vector.
  • a host suitable for the expression vector used when producing the recombinant vector for example, when a bacterium such as E. coli is used as a host, the recombinant vector according to the present invention is capable of autonomous replication in the host, and at the same time, a DNA containing a promoter, an L-arabinitol dehydrogenase gene, and It is preferred to have a configuration necessary for the expression of the transcription termination sequence.
  • PGEX-KG was used as the expression vector used in the present invention, but any expression vector satisfying the above requirements can be used.
  • L-arabinitol dehydrogenase In the production of L-arabinitol dehydrogenase according to the present invention, a transformant obtained by transforming a host with a recombinant vector having a gene encoding the same is cultured, and the gene is cultured (cultured cell or culture supernatant). This is done by producing and accumulating the product L-arabinitol dehydrogenase and obtaining the enzyme from the culture.
  • L-arabinitol dehydrogenase Acquisition and purification of L-arabinitol dehydrogenase is performed by centrifuging the cells or supernatant from the cultures obtained, and by cell alone, affinity chromatography, cation or anion exchange chromatography, or the like. I can do it.
  • the present inventors cloned the gene of L-arabinitol dehydrogenase from Devaromysis hanseni to develop an enzyme capable of producing L-ribulose with high yield. It was confirmed that the recombinant strain incorporating the above-described gene can not only prepare L-ribulose from L-arabitol in high yield, but also greatly reduce the production of by-products, and completed the present invention.
  • the present invention is to clone the gene encoding L- arabinitol dehydrogenase from the gene of devaromysis Hanseni in order to produce industrially useful L- arabinitol dehydrogenase, and from the base sequence of the gene Analyze the inferred amino acid sequence.
  • the L-arabinitol dehydrogenase of the present invention is an enzyme which forms L-ribulose by catalyzing the dehydrogenation reaction using L-arabitol as a substrate, and more preferably has L-arabi specificity for dehydrogenation. L-arabinitol dehydrogenase with the ability to convert toll to L-ribulose.
  • L-arabinitol dehydrogenase of the present invention has the following characteristics: (iii) a molecular weight of about 38 kDa; (Ii) enzymatic activity in the presence of NAD + and Mg 2+ ,
  • L-arabinitol dehydrogenase of the present invention exhibits enzymatic activity in the presence of 1 mM NAD + and 1 mM Mg 2+ .
  • the known L-aravinitol dehydrogenase shows low L-ribulose conversion.
  • the L-arabinitol dehydrogenase of the present invention produces L-ribulose with high yield using L-arabinitol. Therefore, the enzyme of the present invention which produces L-ribulose in high yield in L-arabitol will be very specific and will be usefully applied in the production of L-ribulose from sugar mixtures.
  • FIG. 1 is a diagram illustrating a search through Southern hybridization of a fragment having L-arabinitol dehydrogenase in the devaromysis hanseni chromosome (genomic DNA).
  • No restriction enzyme treatment was performed on the mysis hanseni chromosome (genomic DNA), lanes 2, 3, 4, 5, and 6 were respectively treated with restriction enzymes EcoRI, SalI, BamHI, HindIII, and XbaI.
  • Figure 2 is a vector map of the vector pUC-LAD to find a fragment containing the L- arabinitol dehydrogenase gene on the chromosome of devaromysis Hanseni and cloned into a vector used in E. coli.
  • FIG. 3 is a view showing a method for producing an expression vector comprising the L- arabinitol dehydrogenase gene derived from the devaromysis Hanseni strain.
  • 5 is a diagram showing the appropriate enzyme concentration of L- arabinitol dehydrogenase in the method for producing L-ribulose using the coenzyme solution obtained from the devaromysis Hanseni strain.
  • FIG. 6 is a diagram showing the results of synthesizing L-ribulose using a crude enzyme solution obtained from the devaromysis Hanseni strain.
  • Figure 7 is a diagram showing the production of L-ribulose using the crude enzyme solution obtained from the devaromysis Hanseni strain.
  • the ITS-5.8S rDNA sequence was analyzed at the Korea Microbial Conservation Center. As a result of analyzing the soft relationship with the similar species of the ITS-5.8S rDNA sequence of the S8 strain, it was identified as devaromysis hanseni.
  • the S8 strain was named Debaromyces hansenii KMJ1016, and was deposited internationally in accordance with the Budapest Treaty with Access No. KCCM-10987P on January 30, 2009, to the Korea Microorganism Conservation Center.
  • the yeast bacterium devaromysis hanseni was used to obtain the nucleotide sequence of the L-arabinitol dehydrogenase gene.
  • genes with similar functions are known to be somewhat similar in size to each nucleotide sequence. Therefore, it is estimated that the gene of L-arabinitol dehydrogenase of Devaromysis Hanseni had a size of about 1.0 kb and based on the known L-arabinitol dehydrogenase sequence of other yeasts.
  • the entire gene of L-arabinitol dehydrogenase of Mysis Hanseni was cloned.
  • E. coli XL1-Blue and pUC18 vectors were used for cloning.
  • a culture medium of E. coli LB medium having a general composition was used, and the peptone agar medium (Malt extract peptone agar) was used for culturing devaromysis hanseni.
  • a plate medium of E. coli agar plates containing LB agar, 3-5% sugar, 0.3-0.5% beef extract, 0.9-1.1% bactopeptone, and 1.3-1.7% agar composition were used. 50 ⁇ g / ml ampicillin was added as needed.
  • the culture method was inoculated in a 250 ml Erlenmeyer flask containing 50 ml of devaromysis hanseni and incubated at 37 ° C. and 200 rpm for 1 day, and for E. coli 16 at 37 ° C. and 200 rpm. Time incubation.
  • RNA extraction of devaromysis hanseni was performed using Qiagen plant total RNA kit (QIAGEN), and the reverse transcriptase for cDNA synthesis was Oligo-dT RT-mix (intron).
  • the devaromysis hanseni chromosome was isolated to clone the L-aravinitol dehydrogenase gene.
  • DhLAD F-5 a nonspecific primer based on the L-arabinitol dehydrogenase sequence already known in other yeasts to amplify a portion of the devaromysis hanseni L-arabinitol dehydrogenase gene '-5'- TCA AWB GTR CAG GTM KST GTV GTT CRG MYA TTC AC-3' (SEQ ID NO: 1) and DhLAD R-5'- GAT CAH RMA AGG GAM TTT BYT GGA VCT CKM TAC CAA -3 '(SEQ ID NO: 2) was produced. Using this, a portion of the L-arabinitol dehydrogenase gene corresponding to 730 bp was amplified in the devaromysis hanseni chromosome by a chain poly
  • the genomic DNA of devaromysis hanseni was completely cleaved using restriction enzymes BamHI, EcoRI, HindIII, SalI, and XbaI which do not have a cleavage site in the amplified partial sequences. And a radiolabeled probe was made using a DNA fragment obtained through the polymerase chain reaction. Using this, the DNA fragment containing the gene to be searched by Southern hybridization was searched (FIG. 1). When the chromosome was cut with BamHI, HindIII, and XbaI, the size of the DNA containing the L-arabinitol dehydrogenase gene as a result of Southern hybridization was about 20-23 kb and was not used because it was too large.
  • the desired gene was searched using a fragment cut with EcoRI of about 2.5 kb and SalI of about 5.5 kb.
  • the penicillium pinopylum chromosome was digested with EcoRI, separated from the 2.5 kb DNA fragment and the 5.5 kb DNA fragment isolated from SalI, cloned into pUC18 and named pUC-LAD (FIG. 2).
  • Colony hybridization was performed using the 1.0 kb probe made in the pUC-LAD library to determine clones with the genes of the desired L-arabinitol dehydrogenase.
  • the nucleotide sequence was analyzed using the determined clone, and the total gene nucleotide sequence of L-arabinitol dehydrogenase was found to be 1,056 bp (SEQ ID NO: 3). As expected, it was similar in size to the L-arabinitol dehydrogenase gene found in several other yeasts.
  • Debaromysis Hanseni L-Arabinitol dehydrogenase was found to have the nucleotide sequence common to other L-arabinitol dehydrogenase.
  • the expression vector pGEX-KG (ATCC, USA) BamHI was used to express large amounts of L-arabinitol dehydrogenase in Escherichia coli using a gene encoding L-arabinitol dehydrogenase according to Example 3.
  • the enzyme gene was inserted into the HindIII site and transformed into Escherichia coli BL21 (DE3) (NEB, UK) (FIG. 3).
  • the recombinant strain prepared in Example 4 was inoculated in LB medium and incubated at 37 ° C. for 24 hours, and then the protein expressed in the SDS-PAGE gel was confirmed (FIG. 4).
  • the recombinant strain culture solution was centrifuged (8000 ⁇ g, 10 minutes) to collect only the cells, and then subjected to sonication to the cell wall of E. coli.
  • the supernatant was heat-treated at 70 ° C. for 15 minutes, centrifuged at 20,000 ⁇ g for 20 minutes to remove precipitates, and then the supernatant was obtained.
  • column chromatography was performed using GSTrap (GE Healthcare, Sweden). The recombinant L-arabinitol dehydrogenase was purified purely.
  • Example 5 The physicochemical properties of L-arabinitol dehydrogenase isolated in Example 5 were investigated, and L-arabitol was used as a substrate.
  • the enzyme reaction experiment was carried out under the following conditions.
  • LAD-Arabitol was used as a substrate, and NAD + and NADP + were added as coenzymes.
  • the bacterial culture and enzyme purification were performed in the same manner as in Example 3, and the enzyme and the substrate were reacted at a temperature of 25 ° C. in the presence of 25 mM L-arabitol substrate solution, 1 mM NAD + , and 1 mM NADP + .
  • Example 7 L-ribulose production method using novel L-aravinitol dehydrogenase
  • Example 7-1 Optimum Temperature for L-Riboulose Production
  • Example 5 Production of L-ribulose using L-arabinitol dehydrogenase purely isolated in Example 5 was carried out under the following conditions.
  • the ratio of L-arabitol and L-ribulose was confirmed while changing the temperature.
  • LB As microbial production medium, LB was used.
  • enzyme production medium glycerol 10g L -1 , peptone 1g L -1 , yeast extract 30g L -1 , potassium diphosphate 0.14g L -1 , sodium monophosphate 1g L -1 Added medium was used.
  • absorbance at 600 nm was 0.6
  • 0.1 mM ITPG was added to induce enzyme production.
  • Stirring speed during the process was 200 rpm, the culture temperature was maintained at 37 °C.
  • Example 7-2 Optimal Enzyme Concentration for L-Riboulose Production
  • the L-arabinitol dehydrogenase titration enzyme concentration in L-ribulose production was 15 units ml ⁇ 1 .
  • the production amount of L-ribulose was confirmed while changing the concentration of the initial substrate.
  • Bacterial culture and enzyme purification were performed in the same manner as in Example 3, and the amount of L-ribulose produced was determined using the concentration of L-arabinitol as a substrate in the presence of 1 mM Mg 2+ and 1 mM NAD + as 10-100 g L -1 . Measured. The results are shown in FIG.
  • the productivity of L-ribulose was 57 g L ⁇ 1 h ⁇ 1
  • the conversion of L-ribulose from L-arabitol was 63.1%
  • the production concentration was 63 g L ⁇ 1 .
  • L-ribulose produced by the L-arabinitol dehydrogenase of the present invention is an intermediate of L-ribose synthesis, and L-ribose is L-ribonucleoside, L-oligoribonucleoside and many other treatments. It is an important core pentose that constitutes the skeleton in the synthesis of solvents. The nucleosides derived here have shown considerable potential as useful antiviral agents. Therefore, the conversion of L-arabitol to L-ribulose will play an important role as a basic step for the production of L-ribose.

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Abstract

The present invention relates to L-arabinitol dehydrogenase expressed by the gene of novel L-arabinitol dehydrogenase, to a method for producing L-arabinitol dehydrogenase from strains transformed with a recombinant expression vector containing said gene, and to a method for producing L-ribulose using said dehydrogenase. The recombinant L-arabinitol dehydrogenase of the present invention specifically transforms L-arabinitol only and serves as a stable catalyst for an enzyme reaction. A Debaryomyces hansenii-derived L-arabinitol dehydrogenase and a method for producing L-ribulose using same can be efficiently used in the mass production of L-ribulose. [Representative Drawing] Fig. 5 [Keywords] L-ribulose, Debaryomyces hansenii, L-arabinitol dehydrogenase.

Description

[규칙 제26조에 의한 보정 04.12.2009]　신규 L-아라비니톨 탈수소화효소 및 이를 이용한 L-리불로스의 생산방법 [Correction by Rule 26 04.12.2009] New L-aravinitol dehydrogenase and production method of L-ribulose using same
본 발명은 신규 L-아라비니톨 탈수소화효소 및 그를 이용한 L-리불로스의 제조방법에 관한 것으로서, 보다 상세하게는 L-아라비니톨 탈수소화효소, 이를 코딩하는 핵산 분자, 상기 핵산 분자를 포함하는 벡터, 상기 벡터를 포함하는 형질전환체 및 상기 L-아라비니톨 탈수소화효소를 이용한 L-리불로스의 제조방법에 관한 것이다.The present invention relates to a novel L-arabinitol dehydrogenase and a method for preparing L-ribulose using the same, and more particularly, to L-arabinitol dehydrogenase, a nucleic acid molecule encoding the same, and the nucleic acid molecule. It relates to a vector, a transformant comprising the vector and a method for producing L-ribulose using the L- arabinitol dehydrogenase.
본 발명은 교육과학기술부의 미생물유전체활용기술개발사업의 일환으로 수행된 연구로부터 도출된 것이다[과제 고유번호 : 2007-A002-0065, 과제명: In vitro coevolution에 의한 맞춤형 산화환원효소의 개발].The present invention is derived from a study carried out as part of the microbial genome utilization technology development project of the Ministry of Education, Science and Technology [Task No. 2007-A002-0065, Task name: Development of customized oxidoreductase by in vitro coevolution].
L-리보스(Ribose)는 많은 L-form 핵산당 의약품들의 합성 시작물질이다. L-Ribose는 주로 L-arabinose, L-xylose, D-glucose, D-galactose, D-ribose 또는 D-mannono-1,4- lactone로부터 화학합성법으로 생산되어 왔다. 그러나, 화학합성법은 낮은 총수율, 많은 화학반응 단계, 복잡한 정제과정 및 부산물 형성하는 등 단점이 있다. 이러한 단점을 극복하기 위하여, ribitol 또는 L-ribulose로부터 생물학적 L-ribose 제조법이 연구되고 있다. L-Ribose is the starting material for the synthesis of drugs per many L-form nucleic acids. L-Ribose has been produced mainly by chemical synthesis from L-arabinose, L-xylose, D-glucose, D-galactose, D-ribose or D-mannono-1,4-lactone. However, chemical synthesis has disadvantages such as low total yield, many chemical reaction steps, complex purification process and by-product formation. To overcome this drawback, biological L-ribose preparation from ribitol or L-ribulose has been studied.
최근 의약분야에서 L-탄수화물 및 그의 뉴클레오사이드 유도체의 사용이 크게 증가하고 있는 가운데 특히, 몇 가지의 변형된 뉴클레오사이드는 유용한 항바이러스제로서 상당한 잠재성을 보이고 있다. L-리보오스는 L-리보뉴클레오사이드, L-올리고리보뉴클레오사이드 및 다른 많은 치료용 제재의 합성에 있어서 골격을 구성하는 중요한 핵심 오탄당이다. L-뉴클레오사이드는 D-뉴클레오사이드와 비교할 때, 체내의 뉴클레아제 등의 공격으로부터 높은 안정성을 가지므로 치료용 재료로서 사용될 수 있는 잠재성이 높은 후보 물질이다. L-리보오스가 항바이러스제 및 항암제 등 의약품의 기본이 되는 제조 원료로서 유용성이 높은 것으로 알려지면서 최근에 더욱 주목이 집중되고 있어 L-리보오스의 고효율 생물학적 제조방법의 확립이 요구되고 있다(R. D. Woodyer, N. J. Wymer, F. M. Racine, S. N. Khan, B. C. Saha. Applied and Environmental Microbiology, 2008 74(10):29672975; S. Akiyama,T. Furukawa, T. Sumizawa, Y. Takebayashi, Y. Nakajima, S. Shimaoka, M. Haraguchi. Cancer Science, 2004, 95(11):851857; D. Bou, Y. Kusumanto, C. Meijer, N. H. Mulder, G. Hospers. Pharmacological Research, 2006, 53(2):89-103).In recent years, the use of L-carbohydrates and nucleoside derivatives thereof in the medical field has been greatly increased, in particular, several modified nucleosides have shown considerable potential as useful antiviral agents. L-ribose is an important key pentose sugar that constitutes the backbone in the synthesis of L-ribonucleosides, L-oligoribonucleosides and many other therapeutic agents. L-nucleoside is a high potential candidate that can be used as a therapeutic material because it has high stability against attack of nucleases and the like in the body as compared to D-nucleoside. L-ribose is known to be highly useful as a raw material for manufacturing medicines such as antiviral drugs and anticancer drugs. Recently, more attention has been focused on the establishment of a high-efficiency biological manufacturing method of L-ribose (RD Woodyer, NJ Wymer, FM Racine, SN Khan, BC Saha.Applied and Environmental Microbiology, 2008 74 (10): 29672975 S. Akiyama, T. Furukawa, T. Sumizawa, Y. Takebayashi, Y. Nakajima, S. Shimaoka, M. Haraguchi.Cancer Science, 2004, 95 (11): 851857; D. Bou, Y. Kusumanto, C; Meijer, NH Mulder, G. Hospers.Pharmacological Research, 2006, 53 (2): 89-103).
최근에는 NAD-dependent mannitol-1-dehydrogenase (MDH)를 포함하는 재조합 대장균을 사용하여 100 g/L ribitol로부터 발효 72시간에 55% 전환 수율을 얻었지만 L-ribose의 생산성은 L-arabinose로부터 만드는 화학합성법보다 약 28 배 낮았다(American Institute of Chemical Engineers, W. Ryan, R.Mike, D. David, S. Badal, 2007, Paper No. 11). Klebsiella pneumonia 유래 D-arabinose isomerase, Pseudomonas stutzeri 유래 L-rhamnose isomerase, Streptomyces rubiginosus 유래 D-xylose isomease 및 Lactococcus lactis 유래 galactose-6-phosphate isomerase는 광범위한 기질 특이성을 지녀 L-ribulose를 L-ribose로 전환시킬 수 있지만 그 전환속도는 매우 느리다. 그러나 생산성이 낮고, 또한 L-ribulose로부터 L-ribose의 효소적 생산은 아직 보고되고 있지 않다(Biochimica et Biophysica Acta K. Leang, G. Takada, Y. Fukai, K. Morimoto, T. B. Granstrom, K. Izumori,2004. 1674:68-77). 그러므로 L-ribose 생산성이 높은 경제적인 생물학적인 방법이 개발되어야 한다. Recently, recombinant Escherichia coli containing NAD-dependent mannitol-1-dehydrogenase (MDH) was used to obtain 55% conversion yield at 72 hours of fermentation from 100 g / L ribitol, but the productivity of L-ribose was derived from L-arabinose. About 28 times lower than the synthetic method (American Institute of Chemical Engineers, W. Ryan, R. Mike, D. David, S. Badal, 2007, Paper No. 11). Klebsiella pneumonia-derived D-arabinose isomerase, Pseudomonas stutzeri-derived L-rhamnose isomerase, Streptomyces rubiginosus-derived D-xylose isomease, and Lactococcus lactis-derived galactose-6-phosphate isomerase can convert L-ribulose to L-ribose But the conversion speed is very slow. Low productivity, In addition, enzymatic production of L-ribose from L-ribulose has not yet been reported (Biochimica et Biophysica Acta K. Leang, G. Takada, Y. Fukai, K. Morimoto, TB Granstrom, K. Izumori, 2004. 1674: 68-77). Therefore, economical biological methods with high productivity of L-ribose should be developed.
본 발명에서는 L-아라비톨로부터 L-리불로스를 생산할 수 있는 L-아라비니톨 탈수소화효소 및 그와 같은 효소를 이용하여 L-리불로스를 생산할 수 있는 최적 반응 조건을 제시함으로써, L-리불로스를 높은 수율로 저렴하게 대량 생산할 수 있는 방법을 제공하고자 한다.In the present invention, by presenting L- arabinitol dehydrogenase capable of producing L-ribulose from L-arabitol and the optimum reaction conditions for producing L-ribulose using such enzymes, It is an object of the present invention to provide a method for mass production of low-cost, high-loss products.
본 발명은 상기의 문제점을 해결하고, 상기의 필요성에 의하여 안출된 것으로서 본 발명의 첫 번째 목적은 L-아라비니톨 탈수소화효소의 유전자를 제공하는 것이다.The present invention solves the above problems, and the first object of the present invention is to provide a gene of L-arabinitol dehydrogenase.
본 발명의 두 번째 목적은 상기 유전자로부터 발현된 L-아라비니톨 탈수소화효소를 제공하는 것이다.It is a second object of the present invention to provide an L-arabinitol dehydrogenase expressed from the gene.
본 발명의 세 번째 목적은 상기 L-아라비니톨 탈수소화효소의 유전자를 포함한 재조합 발현벡터를 제공하는 것이다.A third object of the present invention is to provide a recombinant expression vector containing the gene of the L- arabinitol dehydrogenase.
본 발명의 네 번째 목적은 형질전환된 재조합 대장균을 포함하는 모든 형질전환 균주를 제공하는 것이다.It is a fourth object of the present invention to provide all the transforming strains including the transformed recombinant E. coli.
본 발명의 다섯 번째 목적은 형질전환된 재조합 대장균을 이용한 재조합 L-아라비니톨 탈수소화효소를 제공하는 것이다.A fifth object of the present invention is to provide a recombinant L-arabinitol dehydrogenase using transformed recombinant E. coli.
본 발명의 여섯 번째 목적은 상기 효소를 이용하여 L-아라비니톨로부터 L-리불로스를 제조하는 방법을 제공하는 것이다.A sixth object of the present invention is to provide a method for preparing L-ribulose from L-arabinitol using the enzyme.
본 발명의 다른 목적 및 이점은 하기의 발명의 상세한 설명, 청구범위 및 도면에 의해 보다 명확하게 된다.Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.
본 발명은 L-리불로스의 생산방법에 있어서, 데바로마이시스 한세니 (Debaromyces hansenii) 균주의 L-아라비니톨 탈수소화효소를 이용하는 것을 특징으로 한다. 또한 본 발명은 서어던 하이브리다이제이션과 콜로니 혼성화를 통하여 데바로마이시스 한세니로부터 L-아라비니톨 탈수소화효소 유전자를 클로닝하는 것을 특징으로 한다.The present invention is characterized by using the L- arabinitol dehydrogenase of the Debaromyces hansenii strain in the production method of L-ribulose. In another aspect, the present invention is characterized by cloning the L- arabinitol dehydrogenase gene from devaromysis Hanseni through Southern hybridization and colony hybridization.
상기의 목적을 달성하기 위하여 본 발명은 서열번호 4의 아미노산 서열 또는 그 기능적 단편을 가지는 L-아라비니톨 탈수소화효소를 제공한다.In order to achieve the above object, the present invention provides an L-arabinitol dehydrogenase having an amino acid sequence of SEQ ID NO: 4 or a functional fragment thereof.
본 발명의 일 구체예에 있어서 상기 L-아라비니톨 탈수소화효소는 데바로마이시스 한세니에서 유래한 것이 바람직하나 이에 한정되지 아니한다.In one embodiment of the present invention, the L-arabinitol dehydrogenase is preferably derived from debaromysis hanseni, but is not limited thereto.
또한 본 발명의 바람직한 일 구체예에 있어서 상기 아라비니톨 탈수소화효소는 L-아라비니톨에 특이적인 것을 특징으로 한다.In addition, in a preferred embodiment of the present invention, the arabinitol dehydrogenase is characterized in that it is specific to L- arabinitol.
본 발명의 일 구체예에 있어서, 본 발명의 상기 효소의 분자량은 38 kDa인 것을 특징으로 한다.In one embodiment of the present invention, the molecular weight of the enzyme of the present invention is characterized in that 38 kDa.
또한 본 발명의 상기 효소는 1mM Mg2+ 또는 1mM NAD+ 존재 시 활성이 증가하는 것을 특징으로 한다.In addition, the enzyme of the present invention is characterized by an increase in activity in the presence of 1 mM Mg 2+ or 1 mM NAD + .
또한 본 발명은 본 발명의 상기 효소를 코딩하는 L-아라비니톨 탈수소화효소 유전자를 제공한다.The present invention also provides a L- arabinitol dehydrogenase gene encoding the enzyme of the present invention.
본 발명의 상기 유전자는 서열번호 3의 염기서열을 가지는 것이 바람직하나, 유전자 코드의 디제러시 등을 고려하여 서열번호 3에 기재된 서열과 적어도 85% 이상, 바람직하게는 적어도 90% 이상, 더욱 바람직하게는 95% 이상 상동성을 가지는 서열이 바람직하나 이에 한정되지 아니한다.The gene of the present invention preferably has a nucleotide sequence of SEQ ID NO: 3, at least 85%, preferably at least 90% or more, more preferably from the sequence of SEQ ID NO: 3 in consideration of degeneracy of the genetic code, etc. Is preferably a sequence having at least 95% homology, but is not limited thereto.
또한 본 발명은 본 발명의 상기 L-아라비니톨 탈수소화효소 유전자를 포함하는 재조합 발현벡터로 형질전환된 균주를 배양하여 L-아라비니톨 탈수소화효소를 제조하는 방법을 제공한다.In another aspect, the present invention provides a method for producing L- arabinitol dehydrogenase by culturing a strain transformed with a recombinant expression vector comprising the L- arabinitol dehydrogenase gene of the present invention.
또한 본 발명은 본 발명의 상기 L-아라비니톨 탈수소화효소를 이용하여 L-아라비톨로부터 L-리불로스를 제조하는 방법을 제공한다.The present invention also provides a method for preparing L-ribulose from L-arabitol using the L-aravinitol dehydrogenase of the present invention.
이하, 본 발명을 설명한다.Hereinafter, the present invention will be described.
본 발명의 L-아라비니톨 탈수소화효소는 서열번호 4로 표시되는 아미노산서열을 가진 것을 특징으로 한다. 또, 서열번호 4의 아미노산서열에 대해서, 이들 아미노산서열을 가진 단백질이 표시하는 L-아라비니톨 탈수소화효소 활성이 손상되지 않는 범위 내에서, 1이상의 아미노산의 결실, 치환 및 부가의 적어도 1종의 변이가 도입된 변이 L-아라비니톨 탈수소화효소도 본 발명에 관한 L-아라비니톨 탈수소화효소에 포함된다.L-arabinitol dehydrogenase of the present invention is characterized by having an amino acid sequence represented by SEQ ID NO: 4. In addition, at least one amino acid sequence of SEQ ID NO: 4 may be deleted, substituted, or added to one or more amino acids within a range in which the L-arabinitol dehydrogenase activity indicated by the protein having these amino acid sequences is not impaired. Mutant L-arabinitol dehydrogenase into which a mutation of is introduced is also included in the L-arabinitol dehydrogenase according to the present invention.
또, 본 발명에는 서열번호 4의 아미노산서열을 가진 L-아라비니톨 탈수소화효소를 코딩하는 L-아라비니톨 탈수소화효소 유전자가 포함되고, 그 유전자서열로서는 서열번호 3으로 표시되는 것을 들 수 있다. 또, 이들 서열번호 3의 염기서열을 변이시켜서 얻게 되는 상기한 변이 L-아라비니톨 탈수소화효소를 코딩하는 변이 L-아라비니톨 탈수소화효소 유전자도 본 발명에 관한 L-아라비니톨 탈수소화효소 유전자에 포함된다.In addition, the present invention includes an L-arabinitol dehydrogenase gene encoding an L-arabinitol dehydrogenase having an amino acid sequence of SEQ ID NO: 4, and the gene sequence represented by SEQ ID NO: 3 may be mentioned. have. The L-arabinitol dehydrogenase gene encoding the above-mentioned variant L-arabinitol dehydrogenase obtained by mutating the nucleotide sequences of SEQ ID NO: 3 is also L-arabinitol dehydrogenation according to the present invention. It is included in the enzyme gene.
또, 본 발명에는, 상기 L-아라비니톨 탈수소화효소 유전자를 함유하는 재조합벡터, 상기 재조합벡터에 의해서 형질전환된 형질전환체가 포함된다. 또한, 본 발명에는, 이 형질전환체를 배양하여, 얻게 되는 배양물로부터 L-아라비니톨 탈수소화효소를 분리하는 것을 특징으로 하는 L-아라비니톨 탈수소화효소의 제조방법이 포함된다.In addition, the present invention includes a recombinant vector containing the L-arabinitol dehydrogenase gene and a transformant transformed by the recombinant vector. The present invention also includes a method for producing L-arabinitol dehydrogenase, wherein the transformant is cultured to separate L-arabinitol dehydrogenase from the culture obtained.
이하, 본 발명을 더욱 상세히 설명한다. Hereinafter, the present invention will be described in more detail.
본 발명의 L-아라비니톨 탈수소화효소 유전자는 데바로마이시스 한세니의 균체로부터 분리된 것이다. 먼저, L-아라비니톨 탈수소화효소 유전자를 가진 균주로부터 염색체 DNA를 취득한다. L-arabinitol dehydrogenase gene of the present invention is isolated from the cells of devaromysis Hanseni. First, chromosomal DNA is obtained from a strain having a L-arabinitol dehydrogenase gene.
다음에, 설계한 올리고뉴클레오타이드를 프라이머로 하고, 데바로마이시스 한세니 균주의 염색체 DNA를 주형으로 해서 폴리머라제 연쇄반응(PCR)을 행하여, L-아라비니톨 탈수소화효소 유전자를 부분적으로 증폭한다. 이와 같이 해서 얻게 된 PCR 증폭 단편은 데바로마이시스 한세니 균주의 L-아라비니톨 탈수소화효소 유전자에 100% 가까운 상동성을 가진 단편으로서, 콜로니하이브리디제이션을 행할 때의 프로브로서 높은 S/N비를 기대할 수 있는 동시에, 하이브리디제이션의 스트린전시(stringency)제어를 용이하게 한다. 상기의 PCR 증폭 단편을 적당한 시약을 사용해서 표지하고, 상기 염색체 DNA라이브러리에 대해서 콜로니 하이브리디제이션을 행하여, L-아라비니톨 탈수소화효소 유전자를 선발한다(Current Protocols in Molecular Biology, 1권, 603페이지, 1994년). Next, a polymerase chain reaction (PCR) is carried out using the designed oligonucleotide as a primer, and the chromosomal DNA of the devaromysis hanseni strain is used as a template to partially amplify the L-arabinitol dehydrogenase gene. . The PCR amplification fragment thus obtained was a fragment having a homology close to 100% of the L-arabinitol dehydrogenase gene of the devaromysis hanseni strain, and a high S / S probe as a colony hybridization probe. While the N ratio can be expected, it facilitates stringency control of hybridization. The PCR amplification fragments are labeled with appropriate reagents, and colony hybridization is performed on the chromosomal DNA library to select L-arabinitol dehydrogenase genes (Current Protocols in Molecular Biology, Vol. 1, 603). Page, 1994).
상기의 방법에 의해 선발된 대장균으로부터 알칼리법(Current Protocols in Molecular Biology, 1권, 161페이지, 1994년)을 사용해서 플라스미드를 회수함으로써, L-아라비니톨 탈수소화효소 유전자를 함유하는 DNA단편을 얻을 수 있다. 또한, 상기 방법에 의해 염기서열을 결정한 후에는, 상기 염기서열을 가진 DNA단편의 제한효소에 의한 분해에 의해 조제한 DNA단편을 프로브로 해서 하이브리다이즈함으로써 본 발명의 전체 유전자를 얻는 것이 가능하다. 서열번호 3에는 본 발명의 L-아라비니톨 탈수소화효소 유전자의 염기서열을 서열번호 4에는 상기 유전자가 코딩하는 아미노산서열을 표시한다.DNA fragments containing the L-arabinitol dehydrogenase gene were recovered by recovering the plasmid from the Escherichia coli selected by the above method using alkaline protocols (Current Protocols in Molecular Biology, Vol. 1, p. 161, 1994). You can get it. After determining the nucleotide sequence by the above method, it is possible to obtain the entire gene of the present invention by hybridizing the DNA fragment prepared by digestion by restriction enzymes of the DNA fragment having the nucleotide sequence as a probe. SEQ ID NO: 3 shows the nucleotide sequence of the L- arabinitol dehydrogenase gene of the present invention, SEQ ID NO: 4 shows the amino acid sequence encoded by the gene.
본 발명의 형질전환된 미생물은, 본 발명의 재조합벡터를, 상기 재조합벡터를 제작할 때에 사용한 발현벡터에 적합한 숙주 속에 도입함으로써 얻게 된다. 예를 들면 대장균 등의 세균을 숙주로서 사용하는 경우는, 본 발명에 관한 재조합벡터는, 그 자신이 숙주속에서 자율복제 가능한 동시에, 프로모터, L-아라비니톨 탈수소화효소 유전자를 함유하는 DNA 및 전사종결서열 등의 발현에 필요한 구성을 가진 것임이 바람직하다. 본 발명에 사용된 발현벡터로서는 pGEX-KG를 사용하였으나 상기의 요건을 만족하는 발현벡터이면 어느 것이나 사용가능하다.The transformed microorganism of the present invention is obtained by introducing the recombinant vector of the present invention into a host suitable for the expression vector used when producing the recombinant vector. For example, when a bacterium such as E. coli is used as a host, the recombinant vector according to the present invention is capable of autonomous replication in the host, and at the same time, a DNA containing a promoter, an L-arabinitol dehydrogenase gene, and It is preferred to have a configuration necessary for the expression of the transcription termination sequence. PGEX-KG was used as the expression vector used in the present invention, but any expression vector satisfying the above requirements can be used.
본 발명에 관한 L-아라비니톨 탈수소화효소의 제조는, 이것을 코딩하는 유전자를 가진 재조합벡터에 의해 숙주를 형질전환해서 얻은 형질전환체를 배양하고, 배양물(배양균체 또는 배양상청액)속에 유전자 산물인 L-아라비니톨 탈수소화효소를 생성 축적시켜, 배양물로부터 효소를 취득함으로써 행하여진다.In the production of L-arabinitol dehydrogenase according to the present invention, a transformant obtained by transforming a host with a recombinant vector having a gene encoding the same is cultured, and the gene is cultured (cultured cell or culture supernatant). This is done by producing and accumulating the product L-arabinitol dehydrogenase and obtaining the enzyme from the culture.
L-아라비니톨 탈수소화효소의 취득 및 정제는, 얻게 되는 배양물중으로부터, 균체 또는 상청액을 원심 회수하여, 균체파쇄, 친화성크로마토그래피, 양이온 또는 음이온교환크로마토그래피 등을 단독으로 또는 조합함으로써 행할 수 있다. Acquisition and purification of L-arabinitol dehydrogenase is performed by centrifuging the cells or supernatant from the cultures obtained, and by cell alone, affinity chromatography, cation or anion exchange chromatography, or the like. I can do it.
본 발명자는 높은 수율로 L-리불로스를 제조할 수 있는 효소를 개발하고자 데바로마이시스 한세니로부터 L-아라비니톨 탈수소화효소의 유전자를 클로닝하였다. 전기 유전자를 삽입한 재조합 균주가 L-아라비톨로부터 높은 수율로 L-리불로스를 제조할 수 있을 뿐만 아니라, 부산물의 생성을 크게 감소시킬 수 있음을 확인하고, 본 발명을 완성하였다.The present inventors cloned the gene of L-arabinitol dehydrogenase from Devaromysis hanseni to develop an enzyme capable of producing L-ribulose with high yield. It was confirmed that the recombinant strain incorporating the above-described gene can not only prepare L-ribulose from L-arabitol in high yield, but also greatly reduce the production of by-products, and completed the present invention.
본 발명은 산업적으로 유용한 L-아라비니톨 탈수소화효소를 제조하기 위하여 데바로마이시스 한세니의 유전자로부터 L-아라비니톨 탈수소화효소를 암호화하는 유전자를 클로닝하고, 전기 유전자의 염기서열 및 그로부터 유추되는 아미노산 서열을 분석한다. The present invention is to clone the gene encoding L- arabinitol dehydrogenase from the gene of devaromysis Hanseni in order to produce industrially useful L- arabinitol dehydrogenase, and from the base sequence of the gene Analyze the inferred amino acid sequence.
본 발명의 L-아라비니톨 탈수소화효소는 L-아라비톨을 기질로 하여 탈수소화반응을 촉매하여 L-리불로스를 형성하는 효소로서, 보다 바람직하게는 탈수소화에 대한 특이성을 갖고 L-아라비톨을 L-리불로스로 전환시킬 수 있는 능력을 갖는 L-아라비니톨 탈수소화효소를 의미한다.The L-arabinitol dehydrogenase of the present invention is an enzyme which forms L-ribulose by catalyzing the dehydrogenation reaction using L-arabitol as a substrate, and more preferably has L-arabi specificity for dehydrogenation. L-arabinitol dehydrogenase with the ability to convert toll to L-ribulose.
본 발명의 L-아라비니톨 탈수소화효소는 다음의 특징을 갖는다: (ⅰ) 분자량이 약 38kDa; (ⅱ) NAD+와 Mg2+ 존재 시 효소 활성이 나타나고, L-arabinitol dehydrogenase of the present invention has the following characteristics: (iii) a molecular weight of about 38 kDa; (Ii) enzymatic activity in the presence of NAD + and Mg 2+ ,
본 발명의 L-아라비니톨 탈수소화효소는 1mM NAD+와 1mM Mg2+ 존재 시 효소 활성이 나타난다. L-arabinitol dehydrogenase of the present invention exhibits enzymatic activity in the presence of 1 mM NAD + and 1 mM Mg 2+ .
기존에 알려진 L-아라비니톨 탈수소화효소는 낮은 L-리불로스 전환율을 보이고 있다. 그러나 본 발명의 L-아라비니톨 탈수소화효소는 L-아라비니톨을 이용하여 높은 수율로 L-리불로스를 생산한다. 따라서 L-아라비톨에서 L-리불로스를 높은 수율로 생산하는 본 발명의 효소는 매우 특이하다 할 것이며, 당 혼합물로부터 L-리불로스의 생산에 유용하게 적용될 것이다.The known L-aravinitol dehydrogenase shows low L-ribulose conversion. However, the L-arabinitol dehydrogenase of the present invention produces L-ribulose with high yield using L-arabinitol. Therefore, the enzyme of the present invention which produces L-ribulose in high yield in L-arabitol will be very specific and will be usefully applied in the production of L-ribulose from sugar mixtures.
도 1은 데바로마이시스 한세니 염색체(genomic DNA) 내에서 L-아라비니톨 탈수소화효소를 지닌 단편의 서어던 하이브리다이제이션(southern hybridization)을 통한 탐색을 나타낸 도면으로, lane 1은 데바로마이시스 한세니 염색체(genomic DNA)에 아무런 제한효소도 처리하지 않은 것, lane 2, 3, 4, 5, 6은 각각 제한 효소 EcoRI, SalI, BamHI, HindIII, XbaI 등을 처리한 것이다.FIG. 1 is a diagram illustrating a search through Southern hybridization of a fragment having L-arabinitol dehydrogenase in the devaromysis hanseni chromosome (genomic DNA). No restriction enzyme treatment was performed on the mysis hanseni chromosome (genomic DNA), lanes 2, 3, 4, 5, and 6 were respectively treated with restriction enzymes EcoRI, SalI, BamHI, HindIII, and XbaI.
도 2는 벡터 pUC-LAD의 벡터맵으로 데바로마이시스 한세니의 염색체에서 L-아라비니톨 탈수소화효소 유전자를 지니고 있는 단편을 찾아 대장균에서 이용되는 벡터에 클로닝한 것이다. Figure 2 is a vector map of the vector pUC-LAD to find a fragment containing the L- arabinitol dehydrogenase gene on the chromosome of devaromysis Hanseni and cloned into a vector used in E. coli.
도 3은 데바로마이시스 한세니 균주로부터 유래된 L-아라비니톨 탈수소화효소 유전자를 포함하는 발현벡터의 제조방법을 나타내는 도면이다.3 is a view showing a method for producing an expression vector comprising the L- arabinitol dehydrogenase gene derived from the devaromysis Hanseni strain.
도 4는 데바로마이시스 한세니 균주로부터 유래된 L-아라비니톨 탈수소화효소의 SDS-PAGE 젤 사진이다. 4 is a SDS-PAGE gel photograph of L-aravinitol dehydrogenase derived from Devaromysis Hanseni strain.
도 5는 데바로마이시스 한세니 균주로부터 얻은 조효소액을 이용하여 L-리불로스를 생산하는 방법에서의, L-아라비니톨 탈수소화효소의 적정 효소 농도를 나타낸 도면이다.5 is a diagram showing the appropriate enzyme concentration of L- arabinitol dehydrogenase in the method for producing L-ribulose using the coenzyme solution obtained from the devaromysis Hanseni strain.
도 6는 데바로마이시스 한세니 균주로부터 얻은 조효소액을 이용하여 L-리불로스를 합성한 결과를 나타낸 도면이다.6 is a diagram showing the results of synthesizing L-ribulose using a crude enzyme solution obtained from the devaromysis Hanseni strain.
도 7는 데바로마이시스 한세니 균주로부터 얻은 조효소액을 이용하여 L-리불로스의 생산량을 나타낸 도이다.Figure 7 is a diagram showing the production of L-ribulose using the crude enzyme solution obtained from the devaromysis Hanseni strain.
[실시예] EXAMPLE
이하, 본 발명을 다음의 실시예에 의하여 더욱 상세히 설명하나, 본 발명이 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the examples.
실시예 1: L-아라비니톨 탈수소화효소 생산균의 선별Example 1 Screening of L-Arabinitol Dehydrogenase Producing Bacteria
L-아라비니톨 탈수소화효소를 생산하는 균주를 분리하기 위하여 각종 효모균의 배양액 10ul를 생리식염수 10ml에 현탁하고, 현탁액의 10ul(1×104 cfu ml-1)취하여 3% malt extract가 첨가된 펩톤한천배지 (Malt extract peptone agar)에 도말한 후, 37 ℃에서 2일간 배양 하였다. 고체 펩톤배지에서 콜로니가 형성된 후 콜로니를 취하여 L-아라비니톨을 기질로 하여 효소 활성을 가지는 L-아라비니톨 탈수소화효소 활성을 갖는 균들을 선별하는 방법으로 다양한 효모균으로부터 L-아라비니톨 탈수소화효소를 생산하는 버섯균을 탐색하였다.To isolate strains producing L-arabinitol dehydrogenase, 10ul of various yeast cultures were suspended in 10ml of physiological saline, 10ul (1 × 104 cfu ml −1 ) of the suspension was taken, and 3% malt extract added peptone After agar plate (Malt extract peptone agar) was incubated for 2 days at 37 ℃. After colonies were formed in a solid peptone medium, colonies were taken, and L-arabinitol dehydrogenase activity was selected using L-arabinitol as a substrate to dehydrate L-arabinitol from various yeasts. The mushrooms producing digestive enzymes were searched.
위의 탐색과정을 통해 1차 선별된 균주(S1부터 S9까지)를 대조군(C)으로 종래 L-아라비니톨 탈수소화효소 생산균주로 이용되는 트리코더마 리제이를 이용하여, 상기와 같이 L-아라비니톨을 기질로 하여 L-아라비니톨 탈수소화효소 활성이 있는 것을 확인한 후, 효소 활성이 가장 뛰어난 S8 균주를 선별하였다. The first screened strain (S1 to S9) through the above search process as a control (C) using the L- arabinitol dehydrogenase production strain used Trichoderma Reese, as described above, L-arabini After confirming the presence of L-arabinitol dehydrogenase activity using Tol as a substrate, the S8 strain having the highest enzyme activity was selected.
실시예 2: 균주의 동정Example 2: Identification of Strains
실시예 1에서 분리한 S8 균주의 동정을 위하여 한국미생물 보존센터에서 ITS-5.8S rDNA 서열을 분석하였다. 상기 S8 균주의 ITS-5.8S rDNA 서열의 유사종과의 유연관계를 분석한 결과 데바로마이시스 한세니로 동정되었다.     In order to identify the S8 strain isolated in Example 1, the ITS-5.8S rDNA sequence was analyzed at the Korea Microbial Conservation Center. As a result of analyzing the soft relationship with the similar species of the ITS-5.8S rDNA sequence of the S8 strain, it was identified as devaromysis hanseni.
상기 S8 균주는 데바로마이시스 한세니(Debaromyces hansenii) KMJ1016으로 명명하였고, 한국미생물보존센터에 2009년 1월 30일 기탁번호 KCCM-10987P호로로 부다페스트조약에 의거하여 국제 기탁하였다.The S8 strain was named Debaromyces hansenii KMJ1016, and was deposited internationally in accordance with the Budapest Treaty with Access No. KCCM-10987P on January 30, 2009, to the Korea Microorganism Conservation Center.
실시예 3: 데바로마이시스 한세니로부터 신규 L-아라비니톨 탈수소화효소 유전자의 클로닝Example 3: Cloning of a Novel L-Arabinitol Dehydrogenase Gene from Devaromysis Hanseni
L-아라비니톨 분해 효소인 L-아라비니톨 탈수소화효소 유전자의 염기서열을 얻기 위해 효모균 데바로마이시스 한세니를 사용하였다. 일반적으로 유사한 기능을 지니는 유전자의 경우에는 각 염기서열과 크기가 어느 정도 유사하다고 알려져 있다. 따라서 데바로마이시스 한세니의 L-아라비니톨 탈수소화효소의 유전자도 약 1.0kb정도의 크기를 지녔을 것으로 추정하고 다른 효모균의 이미 알려진 L-아라비니톨 탈수소화효소 염기서열을 바탕으로 데바로마이시스 한세니의 L-아라비니톨 탈수소화효소 전체 유전자를 클로닝 하였다.  The yeast bacterium devaromysis hanseni was used to obtain the nucleotide sequence of the L-arabinitol dehydrogenase gene. In general, genes with similar functions are known to be somewhat similar in size to each nucleotide sequence. Therefore, it is estimated that the gene of L-arabinitol dehydrogenase of Devaromysis Hanseni had a size of about 1.0 kb and based on the known L-arabinitol dehydrogenase sequence of other yeasts. The entire gene of L-arabinitol dehydrogenase of Mysis Hanseni was cloned.
클로닝에는 대장균 XL1-Blue와 pUC18 벡터를 사용하였다. 대장균의 배양 배지로는 일반적 조성의 LB 배지를 사용하였고, 데바로마이시스 한세니의 배양에는 상기 펩톤한천배지 (Malt extract peptone agar)를 사용하였다. 대장균의 평판(plate) 배지로는 각각 LB 아가(agar)와 3∼5% 설탕, 0.3∼0.5% 쇠고기 추출물, 0.9∼1.1% 박토 펩톤, 1.3∼1.7% 아가 조성의 아가 플레이트를 사용하였다. 필요에 따라 50 ㎍/ml 엠피실린(amipicillin)을 첨가하였다.  E. coli XL1-Blue and pUC18 vectors were used for cloning. As a culture medium of E. coli, LB medium having a general composition was used, and the peptone agar medium (Malt extract peptone agar) was used for culturing devaromysis hanseni. As a plate medium of E. coli, agar plates containing LB agar, 3-5% sugar, 0.3-0.5% beef extract, 0.9-1.1% bactopeptone, and 1.3-1.7% agar composition were used. 50 μg / ml ampicillin was added as needed.
배양 방법은 데바로마이시스 한세니의 경우, 배지 50 ml이 들어 있는 250 ml의 삼각 플라스크에 접종하여 37℃, 200 rpm 조건에서 1일간 배양하였고, 대장균의 경우에는 37℃, 200 rpm 조건에서 16 시간 배양하였다.  The culture method was inoculated in a 250 ml Erlenmeyer flask containing 50 ml of devaromysis hanseni and incubated at 37 ° C. and 200 rpm for 1 day, and for E. coli 16 at 37 ° C. and 200 rpm. Time incubation.
대부분의 DNA는 아가로스겔(TAE buffer, 0.5%) 전기영동법으로 확인하였고, 겔 상에서 DNA 밴드의 정제는 QiaXII 겔 추출장지(QIAGEN, USA)를 이용하였으며, DNA간의 연결(ligation) 반응은 T4 DNA 연결효소(NEB)를 이용하였다. 또한 데바로마이시스 한세니의 RNA 추출은 Qiagen plant total RNA kit(QIAGEN)을 이용하였으며, cDNA 합성을 위한 역전사 효소는 Oligo-dT RT-mix(intron)를 이용하였다.  Most DNA was identified by agarose gel (TAE buffer, 0.5%) electrophoresis. The purification of DNA bands on the gel was carried out using QiaXII gel extractor (QIAGEN, USA), and the ligation reaction between DNAs was T4 DNA. Ligase (NEB) was used. In addition, RNA extraction of devaromysis hanseni was performed using Qiagen plant total RNA kit (QIAGEN), and the reverse transcriptase for cDNA synthesis was Oligo-dT RT-mix (intron).
L-아라비니톨 탈수소화효소 유전자를 클로닝하기 위하여 데바로마이시스 한세니 염색체를 분리하였다. 데바로마이시스 한세니 L-아라비니톨 탈수소화효소 유전자의 일부분을 증폭하기위해 다른 효모균에서 이미 알려진 L-아라비니톨 탈수소화효소 염기서열을 바탕으로 비특이적 프라이머(degenerated primer), DhLAD F-5'- 5'- TCA AWB GTR CAG GTM KST GTV GTT CRG MYA TTC AC -3' (서열번호 1)와 DhLAD R-5'- GAT CAH RMA AGG GAM TTT BYT GGA VCT CKM TAC CAA -3' (서열번호 2)를 제작하였다. 이를 이용하여 연쇄중합반응에 의해 730bp 크기에 해당하는 L-아라비니톨 탈수소화효소 유전자 일부를 데바로마이시스 한세니 염색체에서 증폭하였다.  The devaromysis hanseni chromosome was isolated to clone the L-aravinitol dehydrogenase gene. DhLAD F-5, a nonspecific primer based on the L-arabinitol dehydrogenase sequence already known in other yeasts to amplify a portion of the devaromysis hanseni L-arabinitol dehydrogenase gene '-5'- TCA AWB GTR CAG GTM KST GTV GTT CRG MYA TTC AC-3' (SEQ ID NO: 1) and DhLAD R-5'- GAT CAH RMA AGG GAM TTT BYT GGA VCT CKM TAC CAA -3 '(SEQ ID NO: 2) was produced. Using this, a portion of the L-arabinitol dehydrogenase gene corresponding to 730 bp was amplified in the devaromysis hanseni chromosome by a chain polymerization reaction.
그리고 증폭된 상기의 부분 염기서열 중 그 절단 부위가 존재하지 않는 제한 효소인 BamHI, EcoRI, HindIII, SalI, XbaI을 이용하여 데바로마이시스 한세니의 genomic DNA를 완전히 절단하였다. 그리고 앞서 중합효소 연쇄반응을 통하여 얻은 DNA 단편을 이용하여 방사능 표지된 탐침자(probe)를 만들었다. 이를 이용하여 서어던 하이브리다이제이션으로 찾고자 하는 유전자를 지닌 DNA 단편을 탐색하였다(도 1). BamHI, HindIII, XbaI으로 염색체를 자른 경우에 있어서는 서어던 하이브리다이제이션의 결과 나타난 L-아라비니톨 탈수소화효소의 유전자를 지닌 DNA의 크기가 약 20∼23 kb정도 되어 너무 큰 관계로 이용하지 않았고, 2.5kb 정도의 EcoRI으로 잘린 조각과 약 5.5 kb정도의 SalI으로 잘린 조각을 이용하여 원하는 유전자를 탐색하였다. 페니실륨 피노필럼 염색체를 EcoRI으로 절단한 후 분리한 2.5kb 정도 크기의 DNA 조각과 SalI으로 절단한 5.5kb 정도의 DNA 단편들을 pUC18에 클로닝하고 이를 pUC-LAD라고 명명하였다(도 2).  The genomic DNA of devaromysis hanseni was completely cleaved using restriction enzymes BamHI, EcoRI, HindIII, SalI, and XbaI which do not have a cleavage site in the amplified partial sequences. And a radiolabeled probe was made using a DNA fragment obtained through the polymerase chain reaction. Using this, the DNA fragment containing the gene to be searched by Southern hybridization was searched (FIG. 1). When the chromosome was cut with BamHI, HindIII, and XbaI, the size of the DNA containing the L-arabinitol dehydrogenase gene as a result of Southern hybridization was about 20-23 kb and was not used because it was too large. , The desired gene was searched using a fragment cut with EcoRI of about 2.5 kb and SalI of about 5.5 kb. The penicillium pinopylum chromosome was digested with EcoRI, separated from the 2.5 kb DNA fragment and the 5.5 kb DNA fragment isolated from SalI, cloned into pUC18 and named pUC-LAD (FIG. 2).
pUC-LAD 라이브러리에서 앞서 만든 1.0kb 크기의 탐침자를 이용하여 콜로니 혼성화를 수행하여 원하는 L-아라비니톨 탈수소화효소의 유전자를 지닌 클론을 결정하였다. 그리고 결정한 클론을 이용하여 염기서열을 분석하여 L-아라비니톨 탈수소화효소의 전체 유전자 염기서열 1,056bp를 밝혔다 (서열번호 3). 이는 앞서 예상한 바와 같이 다른 여러 효모균에서 밝혀진 L-아라비니톨 탈수소화효소 유전자와 크기가 비슷하였다. 또한 데바로마이시스 한세니 L-아라비니톨 탈수소화효소는 다른 L-아라비니톨 탈수소화효소에서 공통적으로 나타나는 염기서열을 지니고 있음을 확인하였다.  Colony hybridization was performed using the 1.0 kb probe made in the pUC-LAD library to determine clones with the genes of the desired L-arabinitol dehydrogenase. The nucleotide sequence was analyzed using the determined clone, and the total gene nucleotide sequence of L-arabinitol dehydrogenase was found to be 1,056 bp (SEQ ID NO: 3). As expected, it was similar in size to the L-arabinitol dehydrogenase gene found in several other yeasts. Debaromysis Hanseni L-Arabinitol dehydrogenase was found to have the nucleotide sequence common to other L-arabinitol dehydrogenase.
또한 진스캔 (http://genes.mit.edu/GENSCAN.html)사이트와 NCBI의 blast 사이트를 이용하여 분석한 결과 인트론 부분이 데바로마이시스 한세니의 L-아라비니톨 탈수소화효소에는 포함되지 않음을 확인하였다. Also, the analysis of gene scan (http://genes.mit.edu/GENSCAN.html) and NCBI's blast site showed that the intron portion was included in L-Arabinitol dehydrogenase of Devaromysis Hanseni. It was confirmed that no.
실시예 4: 재조합 발현 벡터 및 재조합 균주 제조 Example 4: Preparation of Recombinant Expression Vectors and Recombinant Strains
실시예 3에 따른 L-아라비니톨 탈수소화효소를 암호화하는 유전자를 이용하여, 전기 L-아라비니톨 탈수소화효소를 대장균에서 대량으로 발현시키기 위하여, 발현 벡터 pGEX-KG(ATCC, 미국) BamHⅠ과 HindⅢ 부위에 상기 효소 유전자를 삽입한 후 대장균 BL21(DE3)(NEB, 영국)에 형질 전환시켰다 (도 3).The expression vector pGEX-KG (ATCC, USA) BamHI was used to express large amounts of L-arabinitol dehydrogenase in Escherichia coli using a gene encoding L-arabinitol dehydrogenase according to Example 3. The enzyme gene was inserted into the HindIII site and transformed into Escherichia coli BL21 (DE3) (NEB, UK) (FIG. 3).
실시예 5: 재조합 L-아라비니톨 탈수소화효소의 발현 및 순수 분리Example 5 Expression and Pure Separation of Recombinant L-Arabinitol Dehydrogenase
상기 실시예 4에서 제조된 재조합 균주를 LB 배지에 접종하고 37℃에서 24시간 동안 배양한 다음 SDS-PAGE 젤에서 발현된 단백질을 확인하였다 (도 4).The recombinant strain prepared in Example 4 was inoculated in LB medium and incubated at 37 ° C. for 24 hours, and then the protein expressed in the SDS-PAGE gel was confirmed (FIG. 4).
상기 실시예 5의 방법으로 발현시킨 재조합 L-아라비니톨 탈수소화효소효소를 정제하기 위하여, 재조합 균주 배양액을 원심분리 (8000×g, 10분)하여 균체만을 모은 후, 초음파처리하여 대장균의 세포벽을 파쇄하고, 20,000×g에서 20분간 원심분리 하여 침전물(균체)을 제거하고 상등액을 수득하였다. 이어, 상기 상등액을 70℃에서 15분간 열처리하고, 20,000×g에서 20분간 원심분리하여 침전물을 제거한 후, 상등액을 수득한 후, 최종적으로 GSTrap (GE Healthcare, 스웨덴)을 이용한 컬럼 크로마토그래피를 수행하여, 재조합 L-아라비니톨 탈수소화효소를 순수 분리하였다.In order to purify the recombinant L-arabinitol dehydrogenase enzyme expressed by the method of Example 5, the recombinant strain culture solution was centrifuged (8000 × g, 10 minutes) to collect only the cells, and then subjected to sonication to the cell wall of E. coli. Was crushed and centrifuged at 20,000 × g for 20 minutes to remove precipitates (cells) to obtain a supernatant. Subsequently, the supernatant was heat-treated at 70 ° C. for 15 minutes, centrifuged at 20,000 × g for 20 minutes to remove precipitates, and then the supernatant was obtained. Finally, column chromatography was performed using GSTrap (GE Healthcare, Sweden). The recombinant L-arabinitol dehydrogenase was purified purely.
실시예 6: 재조합 L-아라비니톨 탈수소화효소의 특성 실험 Example 6: Characterization of Recombinant L-Arabinitol Dehydrogenase
상기 실시예 5에서 분리된 L-아라비니톨 탈수소화효소의 물리 화학적 특성을 조사하였으며, 기질로서 L-아라비톨을 사용하였다.The physicochemical properties of L-arabinitol dehydrogenase isolated in Example 5 were investigated, and L-arabitol was used as a substrate.
실시예 6-1: 조효소 Example 6-1 Coenzyme
상기 실시예 5의 방법으로 정제한 제조한 L-아라비니톨 탈수소화효소의 조효소를 알아보기 위하여, 효소 반응실험을 다음과 같은 조건에서 수행하였다. L-아라비톨을 기질로 하여 NAD+, NADP+를 조효소로 첨가한 뒤 효소 활성을 측정하였다. 균 배양과 효소 정제 방법은 실시예 3과 같이 수행하였으며, 25mM의 L-아라비톨 기질 용액, 1mM NAD+, 1mM NADP+의 존재 하에서 25℃의 온도 하에서 효소와 기질을 반응시켰다. 표1에 나타난 바와 같이, 1mM NAD+ 존재시 재조한 L-아라비니톨 탈수소화효소의 활성이 나타났으며, NADP+ 존재시에는 그 활성이 나타나지 않았다. 따라서, 본 발명의 L-리불로스 생산 방법에서 L-아라비니톨 탈수소화효소의 조효소로는 1mM NAD+가 필요함을 알 수 있었다.In order to determine the coenzyme of the prepared L- arabinitol dehydrogenase purified by the method of Example 5, the enzyme reaction experiment was carried out under the following conditions. LAD-Arabitol was used as a substrate, and NAD + and NADP + were added as coenzymes. The bacterial culture and enzyme purification were performed in the same manner as in Example 3, and the enzyme and the substrate were reacted at a temperature of 25 ° C. in the presence of 25 mM L-arabitol substrate solution, 1 mM NAD + , and 1 mM NADP + . As shown in Table 1, the activity of the prepared L- arabinitol dehydrogenase in the presence of 1mM NAD + , but not in the presence of NADP + . Therefore, it can be seen that the coenzyme of L-arabinitol dehydrogenase in the production method of L-ribulose of the present invention requires 1 mM NAD + .
표 1
조효소 상대적 활성 (%)(데바로마이시스 한세니의 L-아라비니톨 탈수소화효소)
NAD 100
NADP 6
Table 1
Coenzyme Relative activity (%) (L-Arabinitol dehydrogenase from Devaromysis hanseni)
NAD 100
NADP 6
실시예 6-2: 금속이온의 효과 Example 6-2: Effect of Metal Ions
순수 정제된 L-아라비니톨 탈수소화효소의 금속 이온이 효소 활성에 미치는효과를 알아보기 위하여 본 실험을 수행하였다. 최종농도 1mM의 MgCl2, MnCl2, CoCl2, ZnCl2, 또는 CaCl2를 효소 반응액에 첨가한 후 효소의 잔존 활성을 측정하였다. 1mM 농도에서 다양한 금속의 L-아라비니톨 탈수소화효소 활성에 대한 영향은 표 2에 나타내었다. L-아라비니톨 탈수소화효소는 Mg2+ 존재 시에만 효소 활성이 존재하였다. 따라서, 본 발명의 L-아라비니톨 탈수소화효소의 효소 활성은 1mM Mg2+ 존재 시에만 효소 반응이 일어남을 알 수 있었다.This experiment was performed to investigate the effect of metal ions of pure purified L-arabinitol dehydrogenase on enzyme activity. MgCl 2 , MnCl 2 , CoCl 2 , ZnCl 2 , or CaCl 2 with a final concentration of 1 mM was added to the enzyme reaction solution and the residual activity of the enzyme was measured. The effect of various metals on L-aravinitol dehydrogenase activity at 1 mM concentration is shown in Table 2. L-Arabinitol dehydrogenase had enzymatic activity only in the presence of Mg 2+ . Therefore, the enzyme activity of the L- arabinitol dehydrogenase of the present invention was found to occur only in the presence of 1mM Mg 2+ .
표 2
금속이온 상대적 활성 (%)
1mM
None 2
MgCl2 100
MnCl2 12
CoCl2 7
ZnCl2 4
CaCl2 3
TABLE 2
Metal ion Relative activity (%)
1 mM
None
2
MgCl 2 100
MnCl 2 12
CoCl 2 7
ZnCl 2 4
CaCl 2 3
실시예 7: 신규 L-아라비니톨 탈수소화효소를 이용한 L-리불로스 생산방법Example 7: L-ribulose production method using novel L-aravinitol dehydrogenase
실시예 7-1: L-리불로스 생산을 위한 최적 온도Example 7-1: Optimum Temperature for L-Riboulose Production
상기 실시예 5에서 순수 분리한 L-아라비니톨 탈수소화효소를 이용하여 L-리불로스의 생산실험을 다음과 같은 조건에서 수행하였다. L-아라비니톨 탈수소화효소를 이용하는 본 발명의 L-리불로스 생산방법에서, 온도를 변화시키면서 L-아라비톨과 L-리불로스의 비율을 확인하였다.Production of L-ribulose using L-arabinitol dehydrogenase purely isolated in Example 5 was carried out under the following conditions. In the method for producing L-ribulose of the present invention using L-arabinitol dehydrogenase, the ratio of L-arabitol and L-ribulose was confirmed while changing the temperature.
미생물 생산배지로는 LB를 사용하였고 효소 생산배지로는 글리세롤 10g L-1, 펩톤 1g L-1, 효모 추출물 30g L-1, 이인산칼륨 0.14g L-1, 일인산나트륨 1g L-1가 첨가된 배지를 사용하였다. 600nm에서의 흡광도가 0.6이 될 때, 0.1mM ITPG를 첨가하여 효소 생산을 유도하였다. 상기 과정 중의 교반 속도는 200 rpm, 배양 온도는 37℃로 유지하였다.As microbial production medium, LB was used. As enzyme production medium, glycerol 10g L -1 , peptone 1g L -1 , yeast extract 30g L -1 , potassium diphosphate 0.14g L -1 , sodium monophosphate 1g L -1 Added medium was used. When the absorbance at 600 nm was 0.6, 0.1 mM ITPG was added to induce enzyme production. Stirring speed during the process was 200 rpm, the culture temperature was maintained at 37 ℃.
균 배양과 효소 정제 방법은 실시예 3과 같이 수행하였으며, 1g L-1의 기질 용액, 1mM NAD+, 1mM Mg2+의 존재 하에서 30, 40, 50, 60, 70℃의 온도 하에서 효소와 기질을 반응시켰다. 표2에 나타난 바와 같이, 반응 온도를 50-60℃로 유지하였을 때 우수한 수율을 나타냈으며, 특히 50℃에서 L-리불로스의 생산이 가장 높았다. 따라서, 본 발명의 L-리불로스 생산 방법에서 L-아라비니톨 탈수소화효소와 기질의 반응 시 최적 온도는 50-60℃ 사이임을 알 수 있다.Bacterial culture and enzyme purification were performed in the same manner as in Example 3, in the presence of 1 g L -1 substrate solution, 1 mM NAD + , 1 mM Mg 2+ , enzymes and substrates at temperatures of 30, 40, 50, 60, 70 ° C. Reacted. As shown in Table 2, the yield was excellent when the reaction temperature was maintained at 50-60 ℃, especially the production of L- ribulose at 50 ℃ was the highest. Therefore, in the L-ribulose production method of the present invention it can be seen that the optimum temperature during the reaction of the substrate with L- arabinitol dehydrogenase is 50-60 ℃.
표 3
온도(℃) L-아라비니톨 : L-리불로스
30 45.9 : 54.1
40 41.8 : 58.2
50 36.3: 63.7
60 38.1 : 61.9
70 39.6 : 60.4
TABLE 3
Temperature (℃) L-aravinitol: L-ribulose
30 45.9: 54.1
40 41.8: 58.2
50 36.3: 63.7
60 38.1: 61.9
70 39.6: 60.4
실시예 7-2: L-리불로스 생산을 위한 최적 효소 농도Example 7-2: Optimal Enzyme Concentration for L-Riboulose Production
데바로마이시스 한세니 유래 신규 L-아라비니톨 탈수소화효소를 이용하는 본 발명의 L-리불로스 생산방법에서, L-아라비니톨 탈수소화효소의 적정 효소 농도를 확인하였으며, 그 결과는 도 3에 나타내었다. In the production method of L-ribulose of the present invention using a novel L-arabinitol dehydrogenase derived from devaromysis hanseni, the proper enzyme concentration of L-arabinitol dehydrogenase was confirmed, and the result is shown in FIG. 3. Shown in
도 5에 나타난 바와 같이, L-리불로스 생산에서 L-아라비니톨 탈수소화효소 적정 효소 농도는 15 units ml-1이었다.As shown in FIG. 5, the L-arabinitol dehydrogenase titration enzyme concentration in L-ribulose production was 15 units ml −1 .
실시예 7-3: L-리불로스 생산을 위한 최적 초기 기질 농도Example 7-3 Optimal Initial Substrate Concentration for L-Riboulose Production
L-아라비니톨 탈수소화효소를 이용하는 본 발명의 L-리불로스 생산방법에서, 초기 기질의 농도를 변화시키면서 L-리불로스의 생산량을 확인하였다. 균 배양과 효소 정제 방법은 실시예 3과 같이 수행하였으며, 1mM Mg2+, 1mM NAD+의 존재 하에서 기질인 L-아라비니톨의 농도를 10-100g L-1로 하여 L-리불로스 생성량을 측정하였다. 그 결과는 도 6에 나타내었다.In the L-ribulose production method of the present invention using L-aravinitol dehydrogenase, the production amount of L-ribulose was confirmed while changing the concentration of the initial substrate. Bacterial culture and enzyme purification were performed in the same manner as in Example 3, and the amount of L-ribulose produced was determined using the concentration of L-arabinitol as a substrate in the presence of 1 mM Mg 2+ and 1 mM NAD + as 10-100 g L -1 . Measured. The results are shown in FIG.
도 6에 나타난 바와 같이, L-리불로스 생산에서 기질인 L-아라비톨을 10g L-1로 하였을 때, L-아라비톨에서 L-리불로스로의 변환 비율은 63.5%였고, 기질인 L-아라비톨을 100g L-1로 하였을 때, L-아라비톨에서 L-리불로스로의 변환 비율은 60%였다. 따라서, 최종 농도를 고려하여 본 발명의 L-리불로스 생산 방법에서 초기 기질의 농도는 100g L-1로 하였다. As shown in FIG. 6, when L-arabitol, a substrate in L-ribulose production, was set to 10 g L −1 , the conversion ratio of L-arabitol to L-ribulose was 63.5%, and the substrate L- When arabitol was 100 g L -1 , the conversion ratio of L-arabitol to L-ribulose was 60%. Therefore, in consideration of the final concentration, the concentration of the initial substrate in the L-ribulose production method of the present invention was 100g L -1 .
실시예 7-4: 데바로마이시스 한세니 유래 신규 L-아라비니톨 탈수소화효소를 이용한 최적 조건에서의 L-리불로스 생산Example 7-4 Production of L-ribulose at Optimal Conditions Using Novel L-Arabinitol Dehydrogenase from Devaromysis Hanseni
신규 L-아라비니톨 탈수소화효소를 이용한 최대 L-리불로스 생산을 하기 위하여 상기의 최적 조건에서 이성화 반응을 수행하였다. 100g L-1의 기질 용액, L-아라비니톨 탈수소화효소 15 units ml-1, 50℃, 1mM Mn2+, 1mM NAD+의 존재 하에서 L-리불로스의 생산실험을 수행하였으며, 그 결과는 도 5에 나타내었다.Isomerization was carried out under the optimum conditions for the maximum L-ribulose production using the novel L-aravinitol dehydrogenase. The production experiment of L-ribulose was carried out in the presence of 100 g L -1 substrate solution, L-aravinitol dehydrogenase 15 units ml -1 , 50 ° C, 1 mM Mn 2+ , 1 mM NAD + . 5 is shown.
도 7에 나타난 바와 같이, L-리불로스의 생산성은 57g L-1 h-1, L-아라비톨로부터 L- 리불로스의 전환율은 63.1%, 생산농도는 63g L-1 이었다. 이러한 수율 및 최종 농도는 L-아라비톨부터 L-리불로스 생산에서 매우 우수한 수치이다. 따라서 본 발명의 L-리불로스 생산 방법은 기존의 방법에 비해 우수한 수율 및 고농도로 L-리불로스의 생산이 가능하도록 한다. As shown in FIG. 7, the productivity of L-ribulose was 57 g L −1 h −1 , the conversion of L-ribulose from L-arabitol was 63.1%, and the production concentration was 63 g L −1 . These yields and final concentrations are very good values for L-arabitol to L-ribulose production. Therefore, the production method of L-ribulose of the present invention enables the production of L-ribulose with excellent yield and high concentration compared to the existing method.
본 발명의 L-아라비니톨 탈수소화효소에 의해 생산된 L-리불로스는 L-리보오스 합성의 중간체이고, L-리보오스는 L-리보뉴클레오사이드, L-올리고리보뉴클레오사이드 및 다른 많은 치료용 제재의 합성에 있어서 골격을 구성하는 중요한 핵심 오탄당이다. 여기서 유도된 뉴클레오사이드는 유용한 항바이러스제로서 상당한 잠재성을 보이고 있다. 따라서 L-아라비톨부터 L-리불로스의 전환은 L-리보오스의 생산을 위한 기본 단계로서 중요하게 작용할 것이다.L-ribulose produced by the L-arabinitol dehydrogenase of the present invention is an intermediate of L-ribose synthesis, and L-ribose is L-ribonucleoside, L-oligoribonucleoside and many other treatments. It is an important core pentose that constitutes the skeleton in the synthesis of solvents. The nucleosides derived here have shown considerable potential as useful antiviral agents. Therefore, the conversion of L-arabitol to L-ribulose will play an important role as a basic step for the production of L-ribose.
Figure PCTKR2009000544-appb-I000001
Figure PCTKR2009000544-appb-I000001
[규칙 제26조에 의한 보정 13.04.2009]　
Figure WO-DOC-100
[Revision 13.04.2009 under Rule 26]
Figure WO-DOC-100

Claims (10)

  1. 서열번호 4의 아미노산 서열을 가지는 L-아라비니톨 탈수소화효소.L-aravinitol dehydrogenase having the amino acid sequence of SEQ ID NO: 4.
  2. 제 1항에 있어서, 상기 L-아라비니톨 탈수소화효소는 데바로마이시스 한세니에서 유래한 것을 특징으로 하는 L-아라비니톨 탈수소화효소.The method of claim 1, wherein the L- arabinitol dehydrogenase is L- arabinitol dehydrogenase, characterized in that derived from debaromysis Hanseni.
  3. 제 2항에 있어서, 상기 데바로마이시스 한세니 균주는 데바로마이시스 한세니 (Debaromyces hancenni)(기탁번호 KCCM-10987P)인 L-아라비니톨 탈수소화효소.The L-arabinitol dehydrogenase of claim 2, wherein the debaromysis hanseni strain is Debaromyces hancenni (Accession No. KCCM-10987P).
  4. 제 1항 또는 제2항에 있어서 상기 L-아라비니톨 탈수소화효소는 L-아라비니톨에 특이적인 것을 특징으로 하는 L-아라비니톨 탈수소화효소.The L-arabinitol dehydrogenase according to claim 1 or 2, wherein the L-arabinitol dehydrogenase is specific for L-arabinitol.
  5. 제 1항 또는 제2항에 있어서, 상기 효소는 NAD+ 또는 Mg2+ 존재 시 효소 활성이 나타나는 것을 특징으로 하는 L-아라비니톨 탈수소화효소.The L-arabinitol dehydrogenase according to claim 1 or 2, wherein the enzyme exhibits enzymatic activity in the presence of NAD + or Mg 2+ .
  6. 제1항의 효소를 코딩하는 L-아라비니톨 탈수소화효소 유전자.L- arabinitol dehydrogenase gene encoding the enzyme of claim 1.
  7. 제 6항에 있어서, 상기 유전자는 서열번호 3의 염기서열을 가지는 L-아라비니톨 탈수소화효소 유전자. 7. The L-arabinitol dehydrogenase gene of claim 6, wherein the gene has a nucleotide sequence of SEQ ID NO: 3.
  8. 제 7항의 L-아라비니톨 탈수소화효소 유전자를 포함하는 재조합 발현벡터로 형질전환된 균주를 배양하여 L-아라비니톨 탈수소화효소를 제조하는 방법.A method for preparing L-arabinitol dehydrogenase by culturing a strain transformed with a recombinant expression vector comprising the L-arabinitol dehydrogenase gene of claim 7.
  9. 제 8항에 있어서, 상기 L-아라비니톨 탈수소화효소 유전자는 서열번호 3의 염기서열을 가지는 L-아라비니톨 탈수소화효소를 제조하는 방법.The method of claim 8, wherein the L-arabinitol dehydrogenase gene has a nucleotide sequence of SEQ ID NO: 3.
  10. 제 1항의 L-아라비니톨 탈수소화효소를 이용하여 L-아라비니톨로부터 L-리불로스를 제조하는 방법.A method for preparing L-ribulose from L-arabinitol using the L-arabinitol dehydrogenase of claim 1.
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